The invention relates to a process for preparing baccatin or baccatin derivatives by selective acetylation of the corresponding 10-deacetyl compounds, to an isolated enzyme which catalyses this acetylation reaction and to a process for preparing the enzyme.
Taxol is a promising agent for treating cancer which has antileukaemic and tumour-inhibiting activity (see, for example: M. Suffnes et al., in xe2x80x9cThe Alkaloids, Chemistry and Pharmacologyxe2x80x9d, A. Brossi, Ed., Academic Press: Orlando, Fla., 1985, Vol. XXV, Chapter 1). Originally, taxol was obtained from the bark of certain yew trees (Taxus taxaceae). However, the isolation of taxol from bark is difficult and expensive, and the desired taxol is obtained from the bark in only very poor yields (40 to 165 mg/kg) (see, for example, R. W. Miller et al., J. Org. Chem. 46 (1981) 1469-1474; V. Sxc3xa9nilh et al., J. Nat. Procl. 47 (1984) 131-137; N. Magri et al., J. Org. Chem. 51 (1986) 797-802). Moreover, the use of bark causes the yew trees, which grow back very slowly, to die, so that there are only limited supplies of starting materials.
Since the discovery of the properties of taxol which recommend it for use as a chemotherapeutic agent for cancer, numerous efforts have been made to prepare the compound by synthetic or semi-synthetic processes. Thus, it has been attempted to prepare the taxol structure by organic synthesis (see, for example, W. F. Berkowitz et al., J. Org. Chem. 52 (1987) 1119-1124). However, because of the complexity of the molecule, it has hitherto not been possible to prepare taxol in practically useful amounts by total organic synthesis.
A further route which was used to obtain taxol is partial synthesis starting from a precursor which is easily obtainable in large amounts. One of these routes starts with 10-deacetylbaccatin-III which can be extracted easily and in large amounts from the leaves of Taxus baccata L (G. Chauviere et al., Seances Acad. Sci., Ser. 2, 1981, 293, 501-503). Here, it is possible to isolate approximately 1 g of 10-deacetylbaccatin III per kilogram of leaves, the leaves growing back rapidly. Thus, it is possible without any problems to obtain large amounts of the precursor 10-deacetylbaccatin III.
The desired active compound taxol can be prepared from this precursor, obtained from biological material, by partial synthesis. However, it has been found that, as similar as the structures of 10-deacetylbaccatin III and taxol may be, this partial synthesis still entails significant problems and can for the most part be carried out successfully only by using specific protective groups, giving the desired product taxol in only poor yields.
Denis et al., (J. Am. Chem. Soc. 110 (1988), 5917-5919) describe the synthesis of 10-deacetylbaccatin III to give taxol in two steps. In the first step, 10-deacetylbaccatin III is acetylated chemically in the 10-position. In the second step, baccatin is converted into taxol. However, the first step is not regiospecific, so that acetylation of 10-deacetylbaccatin also occurs, in particular, in position 7. Because of this it is necessary to block the hydroxyl group at this position against acetylation by using a protective group. Exclusive acetylation in the 10-position could only be achieved by using a protective group. However, the use of a protective group entails two more process steps (introduction and removal of the protective group) which is, on the one hand, expensive and, on the other hand, considerably reduces the yield of the product obtained. A further disadvantage of using protective groups consists in the fact that, in particular when the product is used as a pharmaceutical active compound, complicated purification and analysis processes have to be carried out subsequently in order to ensure that there are no more molecules which still carry protective groups present in the product.
Zocher et al. (Biochem. Biophys. Res. Commun., 229 (1996), 16-20) describe a taxol biosynthesis. Here, in an intermediate step, the acetylation of 10-deacetylbaccatin III to give baccatin III was carried out with the aid of crude plant extracts from the roots of Taxus baccata. However, it was not possible to isolate or characterize substances which effect the acetylation. A disadvantage of using a crude extract is the fact that numerous other reactions, in particular acetylation at other positions, can also be initiated or influenced by substances present in the crude extract. Moreover, a crude plant extract has no defined and reproducible composition, so that the use of crude plant extracts results in uncontrollable and varying reactions and yields.
It was therefore an object of the present invention to provide a process for preparing baccatin and baccatin-like baccatin derivatives by selective acetylation of the corresponding 10-deacetyl compounds in position 10. It was a further object to provide an isolated substance which specifically catalyses this reaction.
According to the invention, these objects are achieved by a process for preparing baccatin or baccatin derivatives which is characterized in that 10-deacetylbaccatin or a 10-deacetylbaccatin derivative is reacted in the presence of an isolated enzyme and an acetyl donor, the enzyme being an acetyl transferase having a molecular weight of from 70 to 72 kD, determined by SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis, which acetyl transferase is obtainable from Taxus chinensis cell cultures. It has been found that regioselective acetylation in position 10 is catalysed by an isolated enzyme which can be obtained from suspended Taxus chinensis cell cultures. Surprisingly, it has been found that using the isolated and purified enzyme, it is possible to achieve high regiospecificity with respect to the acetylation in position 10. This specificity is preferably  greater than 80%, more preferably  greater than 90% and most preferably  greater than 95%. It has been found that using the enzyme used according to the invention, it is possible to achieve a specificity of  greater than 99%. Here, a specificity of  greater than 80% means that acetylation has taken place to more than 80% in position 10 and to less than 20% in other positions of the starting material. Consequently, other hydroxyl groups which are present in the starting material do not have to be blocked with a protective group, since acetylation of these other hydroxyl groups occurs to only a very limited extent, if at all, when the enzyme according to the invention is used.
Surprisingly, it has been found that the enzyme used according to the invention has a high substrate specificity. Thus, only 10-deacetylbaccatin or 10-deacetylbaccatin derivatives which have a 10-deacetylbaccatin III-like configuration at and in the vicinity of the C10-position are converted. In particular, baccatin derivatives where the access to position 10 is blocked by voluminous substituents, such as, for example, 10-deacetyltaxol and 10-deacetylcephalomannin, are not acetylated. A precondition for baccatin derivatives to be recognized as substrates by the enzyme according to the invention is therefore that these derivatives, which have a taxane ring structure, essentially correspond to 10-deacetylbaccatin III in positions 7, 8, 9, 10, 11, 12 and 13, i.e. that they do not carry any other substituents in these positions or only substituents having a small volume. The process is preferably suitable for baccatin derivatives which carry the same substituents as 10-deacetylbaccatin III in positions 7 to 13, or carry at least some substituents having a smaller volume than the substituents of 10-deacetylbaccatin III, in particular hydrogen. Voluminous substituents in the other positions do not interfere with the reaction. The process is particularly preferably employed for acetylating 10-deacetylbaccatin III. Furthermore, the process is particularly preferably used for selectively acetylating 14-hydroxy-10-deacetylbaccatin III in position 10.
In contrast, 10-deacetylbaccatin III derivatives whose hydroxyl group in position 7 is blocked by a voluminous protective group, such as, for example, 7-TES-10-DAB III or 7-BOC-10-DAB III, are not recognized as substrates by the enzyme according to the invention. However, such a blocking is not required, since regioselective acetylation in position 10 takes place even when other hydroxyl groups are present in other positions.
Using the process according to the invention, it is possible to acetylate taxane derivatives, which have the same, fewer or less voluminous substituents in positions 7 to 13 than 10-deacetylbaccatin III, selectively in position 10. Such taxane derivatives in which the substituents which are present in 10-deacetylbaccatin III (i.e. OH in position 7, CH3 in position 8, xe2x95x90O in position 9, OH in position 10, CH3 in position 12 and OH in position 13) are present or are replaced by a radical which is smaller or has the same volume, in particular by hydrogen, are included here under the term 10-deacetylbaccatin derivatives, and can likewise be acetylated regiospecifically, if they have an OH group in position 10. Examples of such derivatives are 10-deacetyltaxuyunnanin C, 10,14-deacetyltaxuyunnanin C, 2,10,14-deacetyltaxuyunnanin C, 5,10,14-deacetyltaxuyunnanin C and 2,5,10,14-deacetyltaxuyunnanin C.
The process according to the invention is carried out in the presence of an acetyl donor. Suitable acetyl donors are in principle any substances which donate an acetyl group in the catalytic conversion of the 10-deacetyl starting material. The reaction is preferably carried out in the presence of the acetyl donor acetyl coenzyme A.
From a technical point of view, the use according to the invention of an isolated enzyme offers many advantages. In particular with respect to the conversion rate and with respect to the reproducibility, the reaction can be controlled easily if an isolated enzyme is used.
The enzyme used preferably has an isoelectric point of from pH 5.4 to 5.8, preferably from 5.5 to 5.7 and in particular of pH 5.6. Furthermore, it has been found that the enzyme used according to the invention has a Michaelis constant KM for acetyl coenzyme A of from 55 to 65 xcexcm, preferably of from 59 to 63 xcexcM and in particular of 61 xcexcM.
The invention furthermore provides an isolated enzyme which is characterized in that a) it acetylates 10-deacetylbaccatin III in the presence of an acetyl donor, in particular acetyl coenzyme A, selectively at position 10, b) has a molecular weight of from 70 to 72 kD, determined by SDS-PAGE and c) is obtainable from Taxus chinensis cell cultures.
The enzyme according to the invention is preferably present in a purity of  greater than 50%, in particular  greater than 80%, more preferably  greater than 90% and most preferably  greater than 95%. The enzyme according to the invention is distinguished by the fact that it acetylates 10-deacetylbaccatin III in the presence of an acetyl donor, in particular acetyl CoA, selectively in position 10. This means in particular that acetylation of the other hydroxyl groups of 10-deacetylbaccatin III in positions 1,7 and 13 is virtually not observed. The acetylation reaction has in particular a selectivity of  greater than 50% with respect to position 10, preferably  greater than 80%, more preferably  greater than 90% and most preferably  greater than 95%.
The isolated enzyme is furthermore characterized by a molecular weight of from 70 to 72 kD, determined by SDS-PAGE. To determine the molecular weight, 0.3 xcexcg of homogeneous protein was chromatographed in a denaturizing 10% strength SDS gel in parallel with Marker proteins of a known molecular weight (Rainbow Marker). The proteins were made visible by silver staining, and the molecular weight was determined by comparing the Rf values of the calibration proteins and the enzyme according to the invention. The molecular weight which had been determined by SDS gel electrophoresis was confirmed by gel filtration using a suitable gel filtration column. The gel filtration was carried out using a biosilect-SEC 250-5 column (Biorad) which had been equilibrated using 50 mM tris, pH 8.5, 20 mM 2-mercaptoethanol, in an FPLC unit (Biologic Workstation, Biorad), using a flow rate of 0.2 ml/min. The column was initially calibrated using proteins of a known molecular weight. Under identical conditions, 50 xcexcg of the protein according to the invention were then passed through the column. 250 xcexcl fractions of the eluate were collected, and the activity of the eluate was determined as described below. The molecular weight was determined by comparing the elution times with the known standards.
The enzyme according to the invention can be isolated from cell cultures of Taxus chinensis. It has an isoelectric point of from pH 5.4 to 5.8, preferably from pH 5.5 to 5.7 and in particular of pH 5.6. Furthermore, the Michaelis Menten constant KM found for the enzyme used was from 55 to 65 xcexcM, preferably from 59 to 63 xcexcM and in particular 61 xcexcM for acetyl coenzyme A.
The enzyme according to the invention is an acetyl transferase, in particular an acetyl CoA 10-hydroxytaxane-O-acetyl transferase.
The invention furthermore provides a process for preparing the enzyme described above, which is characterized in that the enzyme is isolated from an enzyme-containing source by using known purification processes and after each purification the fractions in which the enzyme is present are determined by adding 10-deacetylbaccatin or a 10-deacetylbaccatin derivative and an acetyl donor, and the acetylation product formed is detected.
The enzyme-containing source used can be, for example, a plant extract. The enzyme-containing source used is preferably a cell culture, in particular a suspended cell culture. The use of a cell culture is advantageous since it allows large amounts of starting material to be obtained. Compared to using a crude extract as enzyme-containing source, when using a cell culture, it is possible to purify the enzyme to a high degree of purity owing to the large amount of starting material. Particular preference is given to using a starting material originating from Taxus chinensis, for example a Taxus chinensis cell culture.
To purify the enzyme from the starting material, it is possible to employ known purification processes for the isolation of enzymes or proteins. Processes which are preferably used include the ammonium sulphate precipitation from the crude extract, and also chromatographic purification processes, such as, for example, the use of a Sephadex G-25 column, anion exchange chromatography, for example over DEAE-Sephacel, gel filtration, for example over Ultrogel AcA 44, anion exchange chromatography, for example over HighQ, chromatography over a hydroxyapatite column, dye affinity chromatography, for example over High Trap Blue, hydrophobic interaction chromatography, for example over phenyl sepharose and/or dye affinity chromatography, for example over Mimetic Green 1A6XL. Purification preferably includes at least one step in which anion exchange chromatography over HighQ is employed. The HighQ column is an anion exchanger having xe2x80x94N+(CH3)3 groups as ligands. It has been found that in particular in this purification step, substances are removed which catalyse acetylation at positions other than position 10.
In the process according to the invention, the enzyme activity of the fractions is determined after each purification step to determine which fractions contain the enzyme. To this end, the fraction or an aliquot of the fraction is admixed with 10-deacetylbaccatin or a 10-deacetylbaccatin derivative, as defined above, and an acetyl donor. In the fractions in which the desired enzyme is present, product which is acetylated in position 10 can be detected. Preference is given to using 10-deacetylbaccatin III or 10-deacetyltaxuyunnanin C for this test. The acetylation product formed can be detected by using suitable marker groups in the starting materials. Preference is given to using a labelled acetyl donor. Such a labelled acetyl donor comprises a labelled acetyl group which can then be used to determine product which has been acetylated in position 10. Preference is given to using a radioactively labelled acetyl donor. Here, suitable radioactive marker groups are 13C and 14C. The acetyl donor used is particularly preferably an acetyl coenzyme A, in particular [2-14C]-acetyl coenzyme A.
It is also possible to carry out the detection using labelling with a heavy isotope. In this case, the reaction product can be determined by mass spectrometry.
Using the process according to the invention for preparing baccatin or baccatin derivatives employing the enzyme according to the invention, it is possible to prepare baccatin or taxane compounds which have been specifically acetylated in the 10-position. Such compounds are of interest in particular as starting materials for the partial synthesis of taxol. Accordingly, the invention also provides a process for preparing taxol and/or taxol derivatives which is characterized in that baccatin or baccatin derivatives prepared by the process described above are reacted by known processes to give taxol or taxol derivatives. The partial conversion of baccatin or baccatin derivatives to give taxol or taxol derivatives is described in the prior art and entails essentially the introduction of suitable substituents at the hydroxyl group in position 13 of the baccatin derivatives. The baccatin derivatives which are suitable for this purpose consequently have a free OH group at least in position 13.
The reaction of baccatin derivatives to give taxol or taxol derivatives is carried out in particular by esterifying the OH group in position 13 of the baccatin derivatives with a suitable acid. Such processes are described in detail in the literature, for example in U.S. Pat. No. 4,814,470 (Colin et al.,), in U.S. Pat. No. Re. 34,277 (Denis et al.), in EP 0,400,971 A2, in U.S. Pat. No. 4,924,011 (Denis et al.), in U.S. Pat. No. 5,476,954 (Bourzat et al.) and in Denis et al., J. Am. Chem. Soc. 110 (1988), 5917-5919.